Purification and Characterization of a Tungsten-Containing Formate Dehydrogenase from Desulfovibrio gigas
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- 作者：Maria J. Almendra ; Carlos D. Brondino ; Olga Gavel ; Alice S. Pereira ; Pedro Tavares ; Sergey Bursakov ; Rui Duarte ; Jorge Caldeira ; Jose J. G. Moura ; and Isabel Moura
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：December 7, 1999
- 年：1999
- 卷：38
- 期：49
- 页码：16366 - 16372
- 全文大小：81K
- 年卷期：v.38,no.49(December 7, 1999)
- ISSN：1520-4995
文摘
An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formateto carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB9332. D. gigas FDH is a heterodimeric protein [
fchars/alpha.gif" BORDER=0> (92 kDa) and fchars/beta2.gif" BORDER=0 ALIGN="middle"> (29 kDa) subunits] and contains 7 ±1 Fe/protein and 0.9 ± 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrumof D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of1.3 ± 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with twomolybdopterin guanine dinucleotide cofactors. Both Mössbauer spectroscopy performed on D. gigas FDHgrown in a medium enriched with 57Fe and EPR studies performed in the native and fully reduced stateof the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showedthe presence of two signals compatible with an atom in a d1 configuration albeit with an unusual relaxationbehavior as compared to the one generally observed for W(V) ions.
fchars/alpha.gif" BORDER=0> (92 kDa) and fchars/beta2.gif" BORDER=0 ALIGN="middle"> (29 kDa) subunits] and contains 7 ±1 Fe/protein and 0.9 ± 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrumof D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of1.3 ± 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with twomolybdopterin guanine dinucleotide cofactors. Both Mössbauer spectroscopy performed on D. gigas FDHgrown in a medium enriched with 57Fe and EPR studies performed in the native and fully reduced stateof the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showedthe presence of two signals compatible with an atom in a d1 configuration albeit with an unusual relaxationbehavior as compared to the one generally observed for W(V) ions.